结缕草属植物SRAP-PCR体系的建立和优化  

Establishment and optimization of SRAP-PCR reaction system for Zoysia

作　　者：薛丹丹[1,2] 郑轶琦[2] 王志勇[2] 郭海林[2] 陈宣[2] 刘建秀[2] 

机构地区：[1]南京农业大学园艺学院,江苏南京210095 [2]江苏省中国科学院植物研究所,江苏南京210014

出　　处：《草业学报》2008年第6期93-101,共9页Acta Prataculturae Sinica

基　　金：江苏省科技攻关项目(BE2006339);国家自然科学基金项目(30571307);江苏省高技术项目(BG2006320)资助

摘　　要：以SDS法提取的结缕草属植物叶片DNA为模板,分别采用单因子试验和正交设计试验2种方法,对影响结缕草属植物SRAP-PCR的Mg2+、dNTP、引物、Taq DNA聚合酶和模板DNA五个因素进行优化试验。单因子试验分别研究各因素在多水平条件下对SRAP-PCR反应体系的影响,得到最佳反应条件。正交设计采用L16(45)方案,综合考虑各因素间的相互作用,通过直观分析法获得各影响因素的最佳反应水平。根据4对引物对6份结缕草属植物材料扩增结果的验证比较,2种方法所获得的最佳反应体系存在一定的差异。通过综合比较和分析2种方法的优化体系扩增出的条带数、多态性条带数及多态性比率,最终建立了结缕草属植物SRAP-PCR的最佳反应体系:Mg2+2.00 mmol/L、dNTP 220μmol/L、引物0.20μmol/L、Taq DNA聚合酶0.50 U、模板DNA 60 ng、2μL 10×buffer,总体积为20μL。这一优化体系的建立为今后利用SRAP标记技术进行结缕草属植物遗传多样性、种质鉴定、遗传连锁图谱及亲缘关系分析等方面的研究提供了科学的依据。Genetic DNA of Zoysia extracted by the SDS method was used as a template. The major factors of SRAP, such as concentrations of Mg^2＋ , dNTPs, primers, Taq DNA polymerase, and DNA template, were optimized in this study by single factor tests and orthogonal design of five factors at four levels. In single factor tests, the impacts of the SRAP--PCR system were studied for each factor at various levels. In the orthogonal design test, the components of SRAP were considered comprehensively, and a suitable level of each factor was obtained by the intuitional analysis method. Comparing results of four primers and six materials validation, a few differences were found in the amplified electrophoresis of two reaction systems. A comprehensive and deep analysis of the bands, polymorphic bands and polymorphism rate in the two amplified results resulted in an optimised SRAP--PCR system for Zoysia being established： Mg^2＋ 2.0 mmol/L, dNTP 220umol/L, primer 0.2 umol/L, Taq DNA polymerase 0.5 U, DNA template 60 ng, 10×buffer 2uL in the 20 uL volume reaction. The optimized SRAP--PCR reaction system showed that SRAP markers could be widely used for genetic diversity, germplasm identification, construction of genetic linkage maps and relative analysis in Zoysia.

关 键 词：结缕草属植物 SRAP标记 单因子试验 正交设计 体系优化 

分 类 号：S688.4[农业科学—观赏园艺]